This project will elucidate key events in RNA editing and its regulation during the life cycle in T. brucei. The events that occur within the editosome during multiple catalytic steps, movement of mRNA and gRNA to and within the editosome and interactions with these RNAs and editing complexes and proteins will be identified and characterized. The relative roles of the recently discovered compositionally and functionally distinct editosomes in editing of insertion and deletion editing sites will also be investigated. 1) Critical features of editing a single site in vivo will be characterized. Transgenics will be conditionally arrested at specific steps of editing and the compositions and interactions of editosomes and other editing complexes will be compared using a systematic suite of analyses and crosslinking studies. This will illuminate key events in the editing process and generate materials for subsequent aims. 2) Mutants that do not require editing but retain functional editosomes will be analyzed to determine if they have a complete editing system. If not studies will be performed to generate such a cell line by identifying and eliminating editing deficiencies or introducing in normal cells alleles that compensate for the loss of editing. Mutants will also be used to conditionally target RNA to the mitochondrion. These studies will provide invaluable research tools and elucidate the need for editing. 3) Studies that complement those in 1) will characterize functional interactions of editosomes in vitro to identify proteins that bind the mRNA/gRNA anchor duplex, the gRNA 3'oligo (U) tail, insertion vs deletion substrates, and RNA binding that is specific to key catalytic steps. 4) The roles of compositionally and functionally distinct editosomes will be characterized. Turnover of rates of shared and unique proteins will be determined as will be the compositions of editosomes associated with RNAs that differ in editing site composition. Whether proteins exchange between distinct types of editosomes and/or if different types of editosomes physically associate during editing will be assessed as will be the roles in editing of RNA helicases. 5) The basis for the elusive mechanism differential editing during the life cycle will be explored by quantifying edited RNA levels and assessing the relationship between differential gRNA gene content and gRNA sequences in differential editing. The association of gRNAs with editosomes and other editing complexes as well as editosome composition differences between life cycle stages will be characterized. These studies will further the understanding of the roles of the proteins in the editosome and other complexes that function in editing and its regulation. Elucidation of editing processes will provide a basis for the development of drugs for diseases caused by trypanosomatid pathogens. These disease agents all require editing, which is absent in humans. Elucidating the editing process may advance our understanding of other processes such as DNA replication, repair, transcription, reverse transcription and RNA splicing.